Nystatin A was compared with Amphotericin B and Foscarnet for their respective abilities to inhibit the replication of HIV-1 in H9 cells. Nystatin A and Amphotericin B are polyene macrolide antibiotics. Foscarnet is a trisodium phosphonoformate inhibitor of reverse transcriptase (RT). HIV-1 infected cells were cultured for seven days in the presence of 5-50 ug/ml concentrations of these three drugs, and RT activity and p24 antigen production were quantitated. Untreated HIV-1 infected H9 cells served as controls. Nystatin A was most effective inhibiting viral replication at 10 ug/ml, a concentration that did not affect cell viability. Nystatin A treatment inhibited RT activity by 95% and p24 production by 90% which was comparable to the level of inhibition mediated by Amphotericin B and Foscarnet. Western blot analysis of the HIV-infected H9 cells treated with these drugs did not detect any virus at the cellular level. These findings were further corroborated by indirect immunofluorescense studies using monoclonal anti-gp120 antibodies and FITC-conjugated secondary antibodies, and by polymerase chain reaction analysis using a 32P-labelled probe. These results suggest that Nystatin A merits attention as an antiviral drug for the treatment of HIV-1 infection. In vivo drug delivery by liposome encapsulation is currently under study.